Diabetes is a common endocrine and metabolic disease. In recent years, the morbidity of diabetes in the world has been increasing rapidly. With the change of people's life style and quick population aging process in China, the morbidity of diabetes tends to rise rapidly. In 2010, the total number of diabetics in China has been more than 90 million. Diabetes which has become another major noninfectious chronic disease subsequent to cardio-cerebrovascular disease and tumor, severely impairs the health of people. The acute or chronic complications of diabetes, in particular, chronic complications, affect several organs; they have high morbidity and mortality, seriously affect the patient's physical and mental health, and thus bring a heavy burden to individual, family and society.
Insulin therapy is always considered as an important means for treating diabetes and controlling blood sugar suitably. Recently, with the development of the insulin techniques, a new generation of insulin with different acting-time, i.e. insulin analogs, are developed, wherein a newly developed long-acting insulin analog, i.e. insulin glargine, is being accepted and used by more and more physicians and patients.
Recombinant insulin glargine (or recombinant GlyA21-ArgB31-ArgB32-human insulin, or recombinant GlyA21-human insulin-ArgB31-ArgB31-OH) is obtained by mutating asparagine
(Asn) at A21 in chain A of human insulin into glycine (Gly), and adding two arginines (Arg) to the carboxyl terminal of chain B (see: U.S. Pat. No. 5,656,722). The recombinant insulin glargine is able to simulate physiological basis insulin secretion (steady and no peaks), when using it to control the blood sugar to reach the standard level, the patient almost has no risk of low blood sugar, and the pharmaceutical effect can last about 24 hours, which is consistent with work-rest cycle of human life. As a result, it only needs to be injected once per day and is favored by patients and doctors.
In existing recombinant insulin glargine products, most of the recombinant insulin glargines are in the form of solution or amorphous dry powder, without forming crystal. However, the crystal form of recombinant insulin glargine is a better form for application, since it has uniform and steady solid molecular form and small sediment volume, and is easy to be separated from the supernatant, the time for centrifugation and freeze-drying is short, and the production efficiency is relatively high. It is thus desirable to prepare recombinant insulin glargine crystals and then apply the crystals to insulin pharmaceutical preparations.
There are many methods for crystallizing insulin in the prior art, for example, those described in Abel J. J, PNAS, 12: 132(1926), U.S. Pat. No. 2,920,104, CN 95106555.6, etc. However, after extensive research on all kinds of crystallization processes of insulins and analogues thereof, the inventors have found out that the prior methods for crystallizing insulin cannot make the recombinant insulin glargine form crystals: 1) under the conditions for crystallizing human insulin: 0.25M acetic acid, 1.6-2.1 g/L human insulin, 2% zinc, pH 5.95-6.05, insulin glargine cannot form hexahedron crystals, and only exists in the form of amorphous precipitate; 2) under the conditions for crystallizing recombinant Lys-Pro-insulin (LysB28-ProB29-human insulin): 1M acetic acid, 1.8-2.5 g/recombinant Lys-Pro-insulin, 100-300 mg of zinc, 0.2% phenol, pH 5.9-6.2, insulin glargine still cannot form hexahedron crystals, and only exists in the form of amorphous precipitate.
The reason why the prior methods for crystallizing insulin cannot make the recombinant insulin glargine form crystals may lie in that: recombinant insulin glargine has two additional basic amino acids (ArgB31-ArgB32) at the terminal of chain B, which cause its isoelectric point higher than that of natural human insulin, so that under prior conditions for crystallizing insulin, the recombinant insulin glargine can only exist in amorphous state, and cannot form steady hexahedron crystals.